Methods

3. Methods

3.1. Alkaloids

3.1.1. Alkaloid extraction from plant tissues

In a pre-chilled mortar and pestle, 0.5 g plant tissue was ground with liquid nitrogen and

transferred to a 2 ml tube. Alkaloids were extracted with 1 ml 80% (v/v) EtOH with shaking

for 30 min, followed by centrifugation at max speed for 5 min in a bench centrifuge (16,000 x

g). After evaporation of the solvent, the resulting extract was resuspended in 1 ml H2O and

the pH made basic (pH 9.0) with 1 M Na2CO3. Alkaloids were extracted twice with 500 µl

ethylacetate and chloroform and the organic phases evaporated. Finally, the extracts were

resuspended in 100 µl 70% v/v EtOH and analyzed by HPLC and LC-MS.

3.1.2. Analyses by High Performance Liquid Chromatography (HPLC)

HPLC sample analyses was performed using an LC 1100 series Agilent system equipped with

a LiChrospher 60 RP-select B column with a flow rate of 1.0 ml/min and wavelength

detection at 210, 255 and 285 nm. The solvent system and gradient was as follows:

A 98% H2O, 2.0 % CH3CN, 0.01 % H3PO4 Solvent

system B 90% CH3CN, 2.0% H2O, 0.01% H3PO4

Time (min) 0 25 30 35 37 40 43 46 Gradient

% B 0 46 60 60 100 100 0 0

3.1.3. Analyses by Liquid Chromatography- Mass Spectrometry (LC-MS, TOF)

Alkaloids LC-MS analyses were carried out on a Mariner TOF mass spectrometer equipped

with a Turbulon Spray source using an LC 1100 series Agilent system and a Superspher 60

RP-select B column. A flow rate of 0.2 ml/min was used with the following solvent and

gradient system: solvent A CH3CN-H2O (2:98; v/v) and solvent B CH3CN-H2O (98:2; v/v),

0.2% (v/v) formic acid in both solvents. The gradient increased from 0% to 46% B in 25 min,

to 90% in 1 min and was held at 90% for 7 min, post time was 5 min.

I would like to thank Dr. Robert Kramell (IPB) for alkaloid analyses by LC-MS.

16

Methods

3.2. Isolation of RNA

3.2.1. RNA Isolation

Extraction buffer

(EB)

0.8 M guanidine thiocyanate, 0.4 M Ammonium thiocyanate, 0.1 M

Sodium acetate pH 5.0, 5% Glycerol, 38% Phenol (pH 4.5-5.0)

Chloroform, isopropanol, ethanol

Plant tissue-RNA isolation was based on the Trizol® method described by Chomczynski

(1987). In a pre-chilled mortar and pestle, plant tissue was ground with liquid nitrogen and

transferred to a 50 ml conical tube containing extraction buffer EB [10 ml EB/ g tissue],

followed by 1 min vortexing. After incubation for 5 min RT, 2 ml chloroform/ g tissue were

added and the sample mixed vigorously for 20 sec. By centrifugation at 3220 x g for 20 min at

4°C, RNA contained in the aqueous phase was separated from DNA and proteins which

remained in the phenol-chloroform organic phase. One volume isopropanol was added to the

aqueous phase to precipitate the RNA and mixed by inverting the tube gently. The sample was

incubated for 10 min at RT and centrifuged at 3220 x g for 10 min at 4°C. The supernatant

was removed and the RNA pellet washed twice with 70% v/v ethanol with a centrifugation

step in between (3220 x g for 2 min at 4°C). After air drying for 5 min, the pellet was

dissolved in ddH2O. To increase the solubility of the RNA pellet, the tube was heated at 65ºC

for 10 min, followed by 2 min centrifugation in a microcentrifuge at max speed (16,000 x g).

Quality of the RNA was analyzed by gel electrophoresis (3.6.2) and quantified by measuring

the absorbance of a dilute RNA solution at 260 nm (A260). As an absorbance of 1 unit at

260nm corresponds to 40 μg of RNA, the concentration of each sample can be calculated

using the equation:

[RNA] in μg/μl = (40 x dilution factor x absorbance at A260)/ 1000

Purity levels of RNA can be estimated by calculating the ratio between the absorbance value

at 260 nm and 280 nm A260:A280. An acceptable ratio is between 1.5 and 2.0.

17

Methods

3.2.2. Poly-(A)+ RNA isolation

Poly (A)+ RNA was isolated from total RNA obtained from Argemone mexicana roots using

an Oligotex® kit (QIAGEN). This method is based on the hybridization of poly-(A)+ RNA to

oligonucleotides (dC10T30) immobilized on polystyrene-latex particles. In contrast, rRNA and

tRNAs are not polyadenylated and therefore will not bind to the oligo-matrix. The

hybridization of mRNA to the oligotex resin occurs at high salt conditions and is then

recovered from the column with a low salt buffer.

3.3. Isolation of DNA

3.3.1. Plasmid DNA purification

The QIAprep miniprep kit (QIAGEN) was used for the isolation of plasmid DNA from

bacterial cells < 10 kb and a plasmid purification mini Kit (QIAGEN) for plasmid (Bacmid) >

50 kb. This method is based on the alkaline extraction method for plasmid DNA developed by

Birnboim and Doly (1979). Basically, bacterial cells are lysed under alkaline conditions

followed by the neutralization of the lysate in presence of high salt concentrations. In this

way, chromosomal DNA and other contaminants that precipitate are removed by

centrifugation. Plasmid DNA is then absorbed onto a silica membrane, washed and finally

eluted from the column under low-salt concentrations.

3.3.2. Purification of DNA fragments from agarose gel

The MinElute Gel kit (QIAGEN) was used to purify the DNA fragments separated by agarose

gel electrophoresis (3.5.3) following the manufacture’s instructions. To an excised agarose gel

slice, a specified amount of binding buffer was added and the gel slice melted at 50°C. The

molten solution was applied to a column in which the DNA bound to a membrane and

contaminants such as dNTP’s, enzyme and primers were washed out. DNA was eluted from

the column with 10 µl sterile water.

3.3.3. Baculovirus DNA from infected Sf9 cells

Recombinant baculovirus DNA obtained by infection of insect cells was isolated by the

phenol-chloroform method.

18

Methods

Phenol: Chloroform: Isoamylalcohol (25: 24: 1)

3 M sodium acetate pH 5.5

To 300 µl of recombinant viral stock (3.18.6), an equal volume of phenol-chloroform was

added. The sample was vortexed and centrifuged for 1 minute at 10,000 rpm in a bench

centrifuge (9,000 x g). The upper phase was transferred to a new tube and 1/10 volume of 3 M

sodium acetate pH 5.5 (30 µl) and 2 volumes of 100% ethanol (600 µl) were added. The

sample was centrifuged at maximum speed (16,000 x g) for 5 minutes at RT to precipitate the

DNA. The supernatant was removed and the pellet rinsed twice with 80% ethanol. The pellet

was air dried for 5 min and finally resuspended in 50 µl TE buffer or water. One microliter of

the sample was used as a template for a PCR reaction using GSP and vector primers.

3.4. Insect cell culture

3.4.1. Maintenance

Cells of the fall army worm Spodoptera frugiperda Sf9 were routinely incubated at 27ºC in

T-25 flasks containing 5 ml of growth media TC100 medium, 10% v/v foetal bovine serum

(TC100/FBS). After confluence was reached (every 2 days), cells were subcultured 2:1 into a

new flask containing fresh medium. In order to scale up the insect cells cultures, cells were

transferred into a 200 ml conical flask containing 45 ml of TC100/FBS media supplemented

with 0.1% v/v pluronic and incubated at 27°C, 150 rpm,. Cells were subcultured 3:1 after cells

reaches 2x106 cells/ml.

3.5. Electrophoresis

3.5.1. Protein polyacrylamide gel electrophoresis (PAGE)

Destaining solution 30% methanol (v/v), 20% (v/v) glacial acetic acid, water to 1 L

Stain solution 0.2% w/v Coomassie Brilliant Blue R-250 in destaining solution

Running buffer 1X 25 mM Tris base, 250 mM glycine, 0.1% SDS

5X sample buffer 3.12 ml 1 M Tris/HCl (pH 6.8), 1.0 g SDS powder, 2.5 ml glycerol,

75 µl BPB (2% in ethanol), 0.5 µl 2-mercaptoethanol, water to 10 ml

19

Methods

Expressed proteins were analyzed by SDS polyacrylamide gel electrophoresis according to the

protocol described by Sambrook (1989). The components of the separating gel solution (Table

3.1) were mixed and then poured between two glass plates to the desired level. The top of the

gel was overlayed with isopropanol to create a barrier between the gel and the air. The gel was

allow to polymerize 30-60 minutes at RT or until an interface appeared. After removal of the

isopropanol, the stacking gel solution was poured on top of the separating gel in the presence

of a tooth comb (The stacking gel depth was 1/2 well below the deepest level that the comb

teeth reached). After polymerization, the comb was removed and the slab gel was placed on a

vertical electrophoresis chamber. Protein samples were mixed with loading buffer and heated

at 95°C for 5 minutes, then loaded into the wells. A protein molecular weight marker

(Fermentas) was used as reference. The gel ran at 100 V until dye front reached the bottom of

the gel. The protein gel was stained for 2 h and after that destained for 2-3 hours.

Table 3.1 Volume of reagents used for SDS-PAGE Reagent

Separating gel 12% (ml)

Stacking gel 4% (ml)

Acrylamide (30%) 4.0 0.62 1 M Tris/HCl pH=8.8 2.5 ---- 1 M Tris/HCl pH=6.8 ---- 1.25 SDS 10% w/v 0.10 0.05 APS 10% w/v 0.05 0.04 Distilled water 3.30 3.00 TEMED 0.01 0.01

3.5.2. RNA agarose gel

10X MOPS

Denaturing buffer (100 µl)

10x loading buffer

200 mM MOPS, 50 mM Sodium acetate, 10 mM EDTA

pH 7.0 (when autoclaved, it may turn yellow).

13 µl 10X MOPS, 23 µl formaldehyde 37% w/v, 64 µl

formamide, 20 µl 10x loading buffer.

50% v/v glycerol, 0.1 M EDTA pH 8, 0.25% w/v BPB,

0.25% w/v xylenecyanol, 100 µg/ ml EtBr.

The quality of the isolated RNA was assessed by denaturing formaldehyde gel

electrophoresis. For 100 ml of a 1.2% denaturing gel, 1.2 g agarose in 72 ml sterile water was

heated in a microwave oven until completely melted. After cooling the solution to about 60°C,

10 ml 10X MOPS, 5.5 ml formaldehyde 37% w/v and EtBr (0.4 µg/ ml) were added. The

molten gel solution was poured into a tray and allowed to solidify at RT. The samples were

20

Methods

prepared with RNA loading buffer and were denatured for 5 min at 65°C. Electrophoresis was

carried out at 50-80 V in 1X MOPS. RNA was detected by visualization under UV light and

the image recorded using the gel documentation system Gene Genius.

3.5.3. DNA agarose gel

Buffer Working solution Stock solution

TAE 1X

40 mM Tris-acetate,

1 mM EDTA

50X

242 g Tris-base, 57.1 ml acetic acid, 100 ml 0.5 M EDTA

(pH 8.0), volume adjusted to 1 L

DNA fragment analysis was done in a 1% w/v agarose gel with 0.4 µg/ ml EtBr in 1X TAE.

The sample was loaded in 6X DNA loading buffer (Fermentas) and run in 1X TAE at 70-90

V. The size of the DNA fragments were determined by comparison with molecular markers.

Purification of DNA fragments was performed with a MinElute Gel kit (3.3.2 )

3.6. cDNA library

3.6.1. λ-cDNA library construction

A lambda ZAP express cDNA library from Argemone mexicana was constructed by using a

ZAP Express® cDNA Synthesis Kit and mRNA isolated from roots of two-month old plants.

According to the manufacturer’s instructions, the first-strand cDNA was synthesized using

MMLV reverse transcriptase, 7 µg mRNA, oligo(dT) linker-primer containing an XhoI

restriction site and a nucleotide mix containing dATP, dTTP, dGTP and 5-methyl dCTP. 5-

Methyl dCTP hemimethylated cDNA protected the cdNA from digestion with XhoI restriction

enzyme at a later step. Second-strand cDNA started after the addition of Rnase H and DNA

polymerase I. In this step, dCTP was used instead of 5-methyl dCTP to reduce the probability

of 5-methyl dCTP becoming incorporated in the second strand. After second-strand cDNA

synthesis, the uneven termini of the double-stranded cDNA were filled in with Pfu DNA

polymerase. EcoR I adapters as below were ligated to the blunt ends and the double-strand

cDNA was digested with Xho I restriction enzyme. The cDNA was size fractionated by

agarose gel electrophoresis. cDNA fragments less than 400 bp were removed and the

remaining cDNA was ligated into the λ-ZAP Express vector.

5´- AATTCGGCACGAGG-3´

3´- GCCGTGCTCC-5´

21

Methods

Packaging extracts were used to package the recombinant λ-phage following the instructions

of the manufacturer (Gigapack III Gold Packaging Extract; Stratagene). The recombinant

packaged phage was used for titering and library screening.

3.7. Preparation of plating cells for library amplification

One single colony of XL1-Blue MRF’ was inoculated into 20 ml of LB broth (supplemented

with 10 µg/ml tetracycline, 0.2% (w/v) maltose, and 10 mM MgSO4), and incubated at 37ºC

with shaking until an OD600 of 9.0-1.0 was reached. Cells were collected by centrifugation

(1,000 x g, 10 min, 4ºC) and resuspended to an OD600 of 0.5 in 10 mM MgSO4.

3.8. Plating bacteriophage

This procedure was used to isolate pure populations of λ-phage from a single plaque for

screening or providing the titer of the λ-library stock. Following the manufacture’s protocol,

serial dilutions of the packaged phage were prepared in SM buffer. One microliter of the

appropriate serial dilutions was added to 200 µl of prepared plating bacteria (3.7) and

incubated for 15 min at 37°C to allow the phage to attach to the cells. After that, 3 ml of NZY

top agar (melted and cooled to ~ 48°C) supplemented with 7.5 µl 1M IPTG and 50 µl of X-gal

[250 mg/ ml in DMF] were added to the cells and poured onto a LB plate. After the NZY top

agar solidified, the plate was incubated for 8-10 h at 37°C.

3.9. Picking bacteriophage plaques

After recombinant λ virions formed plaques on the lawn of E. coli, each plaque was picked up

using a sterile pipette tip and transferred to a 96-well plate containing 25 µl of SM buffer.

The pipette tip was allowed to stand for 30 min at RT to facilitate bacteriophage diffusion

from the agar into the medium. The phage suspension was used for analysis or stored at -80°C

in SM buffer containing 20% v/v DMSO.

3.10. Library screening

The cDNA insert from a positive recombinant phage was amplified by PCR (3.14.1) using 2

µl of the phage suspension (3.9) and primers T7 and T3. Both primers were complementary to

vector sequences flanking the cloning site. PCR products were analyzed by agarose gel

eletrophoresis (3.5.3) and those cDNA fragments >400 bp were sequenced further (3.14.3).

22

Methods

The resultant cDNA partial sequences were analyzed with the BLAST network service of the

NCBI ( http://www.ncbi.nlm.nih.gov/ ).

Primer Sequence 5’ 3’ Tm T3 GCT CGA AAT TAA CCC TCA CTA AAG 59.3 °C T7 GAA TTG TAA TAC GAC TCA CTA TAG 55.9 °C

3.11. Northern Blot

Northern blotting or northern hybridization is a method developed by Alwine (1977) used for

the analysis of mRNA expression in tissues or cell culture. The steps involved in northern blot

analyzes include:

RNA isolation

RNA fractionation according to size through a denaturing agarose gel

Transfer to a solid support (blotting) and immobilization

Hybridization with DNA or RNA probes

Required solutions for northern blot

Required solution Working solution Stock solution SSC Dehnhardt Prehybridization buffer Wash solution

2X 0.3 M NaCl, 3 mM Sodium citrate 4X SSC, 0.1% SDS, 5X Dehnhardt, 126 µg/ml Salmon sperm 2X SSC with 0.1% SDS

20X 175.3 g NaCl, 88.2 g sodium citrate, adjust the volume to 1 L 100X 2 g BSA, 2 g PVP and 2 g ficoll dissolved in 100 ml water 1 ml 20X SSC, 50 µl 10% SDS, 0.250 ml 100X Dehnhardt, 63 µl Salmon-sperm* (10 mg/ml). Volume adjusted to 5 ml * Heat for 5 min at 95°C, then chill on ice for 5 min.

3.11.1. Blotting

Electrophoresis of total or mRNA was carried out on 1.2% w/v formaldehyde containing gels

as described in section 3.5.2. Subsequently, the gel was soaked in a tray containing 10X SSC

23

Methods

buffer and agitated for 20 minutes to remove the excess formaldehyde. The capillary transfer

system (Figure 3.1) was set up as follows: A piece of Whatman filter paper (3 mm) was

placed over a glass plate placed between two reservoirs filled with 10X SSC; the solution was

allowed to transfer to the paper. All air bubbles between paper and glass were removed by

rolling a pipette across. The gel was placed face down, removing air bubbles between the

paper and gel. A nylon membrane cut to the same size as the gel was soaked in 10X SSC for 5

min and placed on the top of the gel, followed by three pieces of 3 mm Whatman filter paper

presoaked in 10X SSC. Paper towels were stacked over the Whatman paper to absorb the

buffer and the capillary transfer was allowed to proceed overnight. Once completed, the blot

was disassembled and the wells were marked with a pencil. Successful transfer of RNA was

detected by visualization of EtBr stained rRNAs on the membrane under UV light. The

membrane was rinsed in wash solution, and exposed to UV light to crosslink the mRNA to the

membrane. The blot was stored in wash solution at RT or used directly for hybridization.

Figure 3.1. Upward capillary transfer of RNA from agarose gel.

3.11.2. Prehybridization

In a hybridization tube, 5 ml of prehybridization buffer was added to the membrane and

incubated for 3 h at 65°C.

3.11.3. Random primer labelling of DNA

DNA labelling was achieved using a MegaprimeTM DNA Labelling Kit (Amersham).

Basically, random primers are annealed to a denatured DNA template and extended by

Klenow fragment in presence of [α-32P]-dATP. The reaction was set up as follows:

Approximately 50 ng dsDNA in a volume of 21 µl water were denatured by heating to 95°C

for 5 min together with 5 µl of primer mix. Random primer labeling proceded by adding 4 µl

Gel Membrane

Wick

Filter paper Paper towel

24

Methods

each of unlabelled dNTPs (dCTP, dGTP, dTTP), 5 μl [α-32P]-dATP (3000 Ci/mMol) and 2 µl

of the enzyme DNA polymerase 1 Klenow fragment. The content of the tube was gently

mixed and incubated at 37°C for 1 h. Unincorporated nucleotides were removed using a

Sephadex G-50 spin-column.

3.11.4. Hybridization

Labeled DNA was added to the prehybridized membrane and the blot was incubated at 65°C

overnight. Following hybridization, unbound and nonspecifically bound probe were removed

from the membrane by washing 3 times for 15 min at 65°C in wash solution or until getting a

low signal background. The blot was wrapped with Saran wrap and exposed to a

phosphorimager screen overnight. The hybridization incidence was detected with a

Phosphorimager Storm 860.

3.12. First-strand cDNA synthesis

First-strand cDNA synthesis was performed by Moloney Murine Leukemia Virus (MMLV)

reverse transcriptase using mRNA isolated from roots as template.

RACE- cDNA Standard

mRNA 0.5 µg 0.5 µg

5’-oligo(dT)20VN-3’ 12 pmol 200 pmol

BD SMART II A oligo (12 µM) 1.0 µl -

Final volume 10 µl 20 µl

In a microcentrifuge tube, mRNA was denatured for 5 min at 70°C, together with

oligo(dT)20VN, and in the case of 5'-RACE, the BD SMART II A oligo, then chilled on ice.

The reverse transcription reaction was started by addition of 5X reaction buffer, 1µl dNTPs

mix (10 mM each), and 200 U MMLV-RT. The reaction was incubated at 42°C for 1.5 h and

terminated by heating the sample at 72°C for 7 min. Aliquots of the cDNA were stored at

-20°C or used for PCR amplifications. 5'-RACE- cDNA reaction was diluted with 250 µl

Tricine-EDTA Buffer before use or storage.

25

Methods

3.13. Rapid amplification of 5’-cDNA ends (5’-RACE)

According to the kit specifications (BD SMART™ RACE cDNA amplification Kit), two sets

of primers (GSP and NGSP) were designed for 5’-RACE PCR reactions, based on the partial

sequences of the gene of interest. A reactions was composed of 2.5 µl 5'-RACE-Ready cDNA,

5 µl UPM (10X), 1 µl GSP1 (10 µM), 5 µl 10X BD Advantage2 PCR Buffer, 1 µl dNTP Mix

(10 mM), 1 µl 50X BD Advantage2 polymerase mix, 34.5 µl PCR-grade water. The PCR

program was 20 cycles composed of 30 sec denaturation at 94°C, 30 sec annealing at 68ºC

and 3 min extension at 72ºC. PCR products were analyzed by agarose gel electrophoresis

(3.5.3) and positive fragments were cloned into the PCR2.1 vector prior to sequencing. Nested

PCR was performed for those RACE reactions that resulted in a smear of DNA rather than

discrete bands on a gel. The procedure was as above, substituting 5'-RACE-Ready cDNA,

UPM and GSP1 for 5 µl of diluted primary PCR product (1:50 in Tricine-EDTA buffer), 1 µl

of the NUP primer and 1 µl of NGSP using the same PCR program as above.

3.14. Polymerase chain reaction (PCR)

3.14.1. Standard PCR reaction

The Polymerase Chain Reaction method developed by Mullis and Faloona (1987) is a cycling

reaction in which a specific DNA fragment is denatured by heating to separate dsDNA,

followed by hybridization of the denatured DNA with specific primers (annealing). The cycle

ends as the primer molecules are elongated by the action of DNA polymerase to produce a

new strand of DNA. Repeating the cycle (usually 25 to 35 times) produces sufficient amounts

of the specific DNA fragment for analyzes. A standard PCR reaction was set up as below:

PCR mix amount

10X PCR reaction buffer

dNTP’s (10 mM)

5’ and 3’ primer (10 µM)

DNA template1

DNA polymerase2

H2O

3.0 µl

0.5 µl

2.0 µl

100-200 ng**

0.5 U

adjust to 30 µl 1 For full-length amplifications, 2 μl cDNA (3.12) was

used as template.

26

Methods

Thermal cycling conditions

Step Temp (°C)

Time cycles

Initial denaturation

95

1-3 min 1

Denaturation 95 0.5-2 min Annealing 45-65 30 sec Extension 72 2-4 min

25-35

Final extension 72 5 min 1 Stop 4

2 Taq polymerase was used for routine PCR reactions

and Pfu polymerase was used for full-length

amplifications.

3.14.2. Screening bacterial colonies by PCR

This method was used as a preliminary step to identify bacterial colonies containing

recombinant plasmid. Using a white tip, a single colony at random (or white in blue/white

screening) was picked up and streaked onto a fresh LB plate used as a replica (about 20

colonies on a single plate). The plate was incubated at 37°C, and then stored at 4°C until

needed. After streaked onto a plate, the tip was then dipped into a PCR tube containing PCR

mix and a PCR reaction was run as described in (3.14.1) using vector primers or GSP. The

amplified PCR fragments were analyzed by agarose gel electrophoresis (3.5.3). Colonies

yielding products of the expected size were selected for plasmid isolation (3.3.1) and DNA

sequencing.

3.14.3. Sequencing of DNA

Nucleotides sequences were determined using a BigDye™Terminator cycle sequencing kit in

an automated DNA sequencer ABI 3100 Avant Genetic Analyzer (Applied Biosystems). The

standard reaction conditions were as follows:

Sequencing mix Volume ( µl) BigDyeMix V 1.1 Primer (5 µM) Plasmid-DNA** H2O

4.0 1.0 1-5

Adjust to 10 µl ** 600 ng plasmid; 20 ng PCR product

27

Methods

Cycle sequencing Step Temp (°C) Time

25 cycles Denaturation Annealing Elongation

96 50 60

10 sec 5 sec 4 min

Stop 4

Once the sequencing reaction was completed, samples were purified by gel filtration with

sephadex G-50 superfine (20-80µm) columns.

Sequences were compared using the BLAST program provided by the NCBI

(http://www.ncbi.nlm.nih.gov/BLAST/).

3.15. DNA modifications

3.15.1. Addition of a 3’-A Overhang

Pfu DNA polymerase generates blunt-ended PCR products due to its 3’ and 5’ exonuclease

proofreading activity that removes the 3’ overhangs necessary for TA cloning. However 3’-A

overhang can be added to blunt-end fragments using Taq DNA polymerase after PCR

amplifications. In a vial on ice, 7 µl of PCR reaction were mixed with 0.2 unit of Taq

polymerase, 2 µl of dATP 1 µM and 1 µl 10X Taq Buffer and incubated at 72°C for 15

minutes. After agarose gel purification (3.3.2), fragments were ligated into a T-vector

(3.16.1).

3.15.2. Dephosphorylation of DNA fragments

In order to prevent self-enclosure of a vector in ligation reactions, those vectors digested with

a single restriction enzyme where dephosphorylated (hydrolysis of the 5´-terminal phosphate

residue) using calf intestine alkaline phosphatase (CIAP) enzyme. After vector digestion with

a restriction endonuclease, 3 µl of 10X buffer, 1 µl CIAP were added to the reaction and the

volume adjusted to 30 µl with sterile water. The sample was incubated for 30 min at 37°C

followed by DNA purification on agarose gel (3.3.2).

28

Methods

3.15.3. Restriction enzyme digestion

Restriction endonucleases recognize specific sequences in the DNA and cleave a

phosphodiester bond on each strand at that sequence. A general procedure for conducting

restriction digestion consisted of 1X reaction buffer, 1X BSA (if required), 0.5-1.0 µg DNA,

and 1 U enzyme per µg DNA in a total volume of 20 µl. The sample was incubated at 37°C

for 3 hr and heat inactivated before adjusting conditions for a second enzyme (if required).

The resultant DNA fragments were separated by agarose gel electrophoresis (3.5.3) and

purified using a minelute kit (3.3.2).

3.16. DNA cloning

The ligation of a DNA fragment into a vector was catalyzed by T4 DNA ligase. This enzyme

catalyzes the formation of a phosphodiester bond by the condensation of a 5' phosphate and 3'

hydroxyl group of adjacent nucleotides occurring in a nick or between cohesive or blunt

termini of DNA.

3.16.1. TA cloning

PCR products with a 3’-A overhang can be directly cloned into a linearized vector with a

complementary 3’-T overhang. The amount of PCR product needed to ligate with 50 ng

T-vector (pCR2.1 or pGEM-T easy) was calculated as below. The ligation reaction was set

up by mixing the calculated fresh PCR product, 1X ligation buffer, 50 ng vector, 1 µl of the

T4 DNA ligase (3-4 Weiss units/ µl) in a total volume of 10 µl. The sample was briefly

centrifuged and incubated overnight at 14°C. The recombinant plasmid was transformed into

the appropriate competent cells and purified for further subcloning or sequencing. A ratio 3:1

insert to vector was normally used.

insert (ng of vector) (kb size of insert)

3.16.2. Subcloning

The gene to be expressed in Sf9 insect cells was first excised from a T-vector with specific

restriction enzymes (3.15.3) and inserted into the same sites of a baculovirus transfer vector

kb size of vector * molar ratio ng of insert =

vector

29

Methods

(pVL1392 or pFastBac1) pre-digested and dephosphorylated (if required). Both vectors

contain a multiple cloning site downstream of a baculovirus promoter required for expression

of proteins in insect cells. The amount of insert and the conditions required to ligate with 100

ng transfer vector were calculated as in (3.16.1). The reaction was carried out at 4°C overnight

in a 20 µl ligation mixture containing 2μl 10X ligase buffer, 1 μl T4 DNA ligase (3 U/μl), 100

ng Vector and the insert.

3.17. Transformation of competent cells

Once the ligation reaction was completed, a standard procedure for transformation of bacteria

was performed as follows: In an Eppendorf tube on ice, 2 µl of ligation product were added to

50 µl competent cells and left on ice for 30 min. After that, cells were heat shocked for 30 sec

in a water bath at 42°C and placed back on ice for 2 min. After addition of 250 µl SOC

medium, cells were grown at 37°C for 1 h with shaking (225 rpm). Twenty to 100 µl from

each transformation were plated onto LB plates containing the appropriate supplement

(below) and incubated overnight at 37°C.

Supplements required for LB plates

Cells/vector Supplements

TOP10 cells/pCR2.1 50 µg/ ml kanamycin

XL1-Blue MRF’ cells/ pGEM-T 100 µg/ ml ampicillin, 0.5M IPTG, 80 µg/ ml X-Gal

After transformation, plasmid was purified and analyzed for correct insertion of the gene by

restriction endonuclease digestion (3.15.3), PCR analysis (3.14.1) or sequencing (3.14.3).

3.18. Protein Expression

3.18.1. BaculoGold Expression Vector System

The Baculovirus Expression Vector System (BEVS) from BD-PharMingen uses the

Autographa californica nuclear polyhedrosis virus (AcNPV) for the expression of foreign

genes in insect cells. The gene of interest is cloned in a transfer vector within flanking sites

homologous to the AcNPV DNA. Recombination between both sites occurs via co-

transfection of the transfer vector and the AcNPV DNA into Spodoptera frugiperda (Sf) cells.

30

Methods

Infection and amplification of Sf9 cells with the recombinant virus results in the expression of

mRNA and protein production.

3.18.2. Co-transfection using BD Baculogold

Transfection buffer A: Grace's medium with 10% FBS

Transfection buffer B: 25 mM HEPES pH 7,1; 125 mM CaCl2 ; 140 mM NaCl

Recombinant baculovirus was generated by cotransfection of linearized baculovirus DNA

(BD Baculogold) and pVL1392 vector containing the gene of interest by using BD

Baculogold kit. Co-transfection in insect cells was developed as follows:

(1) 5x105 Sf9 cells/well was seeded onto a 37 mm 6 well plate. After 15 minutes, the

culture medium was removed and replaced with 1 ml of transfection buffer A.

(2) In a sterile Eppendorf tube, 0.05 µg baculovirus DNA was mixed with 2 µg of

recombinant plasmid and after 5 min, 1 ml of transfection buffer B was added.

The mixture from the step 2 containing buffer B was added drop-by-drop to the insect cells in

the step 1, rocking the plate back and forth to mix the newly added solution with the

transfection buffer A. The plate was incubated at 27°C for 4 hr after that, the solution was

removed and cells were washed with 3 ml of TC-100 medium. Finally, 3 ml of TC100/FBS

medium were added to the plates and incubated at 27°C for 5 days. After 5 days the medium

was harvested and centrifuge (700 x g) for 5 min at RT. The supernatant containing

recombinant virus was used for further rounds of amplification (3.18.6) or stored at 4ºC in a

dark place.

3.18.3. BAC-to-BAC expression system

In the baculovirus expression system from Invitrogen, the gene of interest is cloned into a

transfer vector (pFastBac1) and then inserted into a bacmid (baculovirus shuttle vector)

propagated in E. coli cells (DH10Bac) by site-specific transposition. Bacterial colonies

containing the recombinant bacmid DNA can be selected by white/blue selection and the

purified recombinant bacmid DNA used for transfection in insect cells.

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Methods

3.18.4. Bacmid transposition

Recombinant plasmid (pFastBac1) was transformed into DH10Bac competent cells by

mixing 3 µl of ligation mix with 40 µl of the cells. The sample was incubated on ice for 30

min, heat shocked for 45 sec in a water bath at 42°C and placed back on ice for 2 min. After

addition of 900 µl SOC medium, cells were grown at 37°C for 4 h with gentle shaking. Fifty

to 100 µl were plated onto LB plates containing 50 µg/ ml kanamycin, 7 µg/ ml gentamycin,

10 µg/ ml gentamycin, 10 µg/ ml tetracycline, 80 µg/ ml X-Gal, 40 µg/ ml IPTG and

incubated for 24 to 48 h at 37°C. White colonies were selected for recombinant bacmid DNA

isolation (3.3.1). The insertion of the gene of interest into the bacmid was confirmed by PCR

(3.14.1) using GSP and vector primers.

3.18.5. Transfection of Sf9 insect cells with recombinant bacmid DNA

Transfection of recombinant bacmid was performed using the Cellfectin reagent (Invitrogen)

according to the manufacturer’s instructions. The procedure was as follows: In a 6-well tissue

culture plate, 5 x 105 Sf9 cells/well were seeded and allowed to attach to the bottom of the

plate for 30 min at 27°C, then washed once with 2 ml of TC100 medium. For each

transfection in insect cells, 5 µl of mini-prep bacmid DNA and 6 µl Cellfectin (lipid reagent)

were diluted separately into 100 µl TC100 Medium. Both solutions were mixed together and

incubated for 40 min at RT, then diluted to 1 ml with TC100 medium and added to the cells.

The cells were incubated for 5 h at 27°C after which the transfection medium was removed

and replaced with 2 ml TC100/FBS. Cells were incubated for 3-4 days at 27°C. The

supernatant containing recombinant virus was used for further rounds of amplification

(3.18.6) or stored at 4ºC protected from light.

3.18.6. Baculovirus amplification

The recombinant baculovirus obtained after transfection of Sf9 cells (3.19.2 or 3.19.5), was

further amplified three times by infecting additional Sf9 insect cells. 1 ml of the previous

amplified virus was added to a T-25 flask containing 2.0 x 106 cells/ml in TC100/FBS, and

incubated at 27°C for 4 or 5 days. After about 90% of the cells were lysed, the supernatant

was harvested from the plate and the cellular debris spin down in a centrifuge at 700 x g; 5

min at RT. Supernatant was transferred into a sterile tube and stored at 4°C in a dark area. A

high recombinant virus stock was preparing by infecting 50 ml insect cell culture

supplemented with TC100/FBS/P, with the resultant 5 ml recombinant virus from the fourth

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Methods

amplification. Cells were incubated for 4 to 5 days at 27°C with shaking (150 rpm) until about

90% of the cells were lysed. After that, cells were centrifuge at 700 x g for 5 min at RT and

the supernatant containing virus stored at 4°C in a dark area. This viral stock was used to

overexpress the protein of interest in insect cells.

3.18.7. CYP450 expression in Sf9 cells

Resuspension buffer 100 mM Tricine/NaOH pH 8.0, 5 mM TGA, 10% v/v

glycerol, 2 mM EDTA, 10 mM 2-mercaptoethanol.

In a 200 ml conical flask, 50 ml Sf9 cell cultures were grown to a density of 2.0 x 106 cells/ml

in TC100/FBS/P medium and recovered by centrifugation at 700 x g for 5 min at RT. Cells

were resuspended in 10 ml of TC100/FBS/P medium and mixed with 0.5 ml recombinant

P450 viral stock and 0.5 ml of a viral stock containing cDNA that encodes NADPH-

cytochrome P450 reductase (Huang 2000). Sf9 cells were incubated at 27C for 1 h with

shaking at 150 rpm, after that, 40 ml of TC100/FBS/P medium were added. To compensate

for the low endogenous level of hemin in insect cells, hemin was added to a final

concentration of 2 µg/ml 24 h after infection. Sf9 cells were harvested 72 h after infection or

until the cells enlarged and about 10-20% of cells were disrupted. Infected cells were

collected by centrifugation (700 x g, 5 min, RT) and resuspended in 3.5 ml of resuspension

buffer.

3.18.8. Ctg9 and ctg11 expression in Sf9 cells

Resuspension buffer

A) 100 mM Tricine/NaOH pH 7.5, 5 mM TGA, 10% v/v

glycerol, 2 mM EDTA, 10 mM 2-mercaptoethanol.

B) 50 mM phosphate buffer pH 7.5 or 9.0

C) 50 mM phosphate buffer pH 7.5 or 9.0, 1.0 % emulgen 913

In a 200 ml conical flask, 50 ml Sf9 cell cultures were grown to a density of 2.0 x 106 cells/ml

in TC100/FBS/P medium and recovered by centrifugation at 700 x g for 5 min at RT. Cells

were resuspended in 10 ml of TC100/FBS/P medium and mixed with 0.5 ml recombinant ctg9

or ctg11 stock. Sf9 cells were incubated at 27C for 1 hr with shaking at 150 rpm, after that,

40 ml of TC100/FBS/P medium were added. Sf9 cells were harvested 72 h after infection or

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Methods

until the cells enlarged and about 10-20% of cells were disrupted. Infected cells were

collected by centrifugation (700 x g, 5 min, RT) and resuspended in 3.0 ml of resuspension

buffer.

3.19. Enzymatic assay

3.19.1. CYP450 activity assays

The standard conditions for activity assays consisted of 60 µl cell suspension (3.18.7), 200

mM tricine/NaOH pH 8.0, 0.5 mM NADPH and 50 µM substrate in a total volume of 80 µl.

The assay mixture was incubated for 30 min and then terminated by the addition of 1 volume

methanol. After protein precipitation, the sample was centrifuge at maximum speed for 2 min

in a centrifuge bench. The reaction products were analyzed by HPLC and LC-MS (3.1.2 and

3.1.3). For pH studies, the buffer was phosphate buffer pH 6.0 – 8.5, tricine buffer pH 7.4 –

8.8, glycine buffer pH 9.0 – 10.0.

3.19.2. Ctg9 and Ctg11 activity assays

The standard conditions for activity assays consisted of 150 µl cell suspension or supernatant

(3.18.8) with 100 mM tricine/NaOH (pH 7.5) or 100 mM phosphate buffer (pH 7.5 or 9.0) and

50 µM substrate in a total volume of 200 µl. The assay mixture was incubated for 2 h at 30

and 37C and then terminated by the addition of 1 volume methanol. After protein

precipitation, the sample was centrifuge at maximum speed for 2 min in a centrifuge bench.

The samples were concentrated and then analyzed by HPLC and LC-MS (3.1.2 and 3.1.3).

3.20. Preparation of microsomes

500 ml of Sf9 suspension culture was grown to a density of 2x106 cells/ml in TC100/FBS/P

medium and collected by centrifugation at 700 x g for 5 min. The pellet was resuspended in

100 ml fresh medium, followed by the addition of 6.5 ml of recombinant P450 viral stock.

After 1 h incubation at 27°C and 150 rpm, 400 ml TC100/FBS/P medium was added and the

suspension incubated for 72 h. Hemin was added to a final concentration of 2 µg/ml 24 h after

infection. Cells were collected by centrifugation (1000 x g, 5 min, RT) and the pellet washed

twice with 40 ml PBS buffer (130 mM NaCl, 7 mM Na2HPO4, 3 mM NaH2PO4, pH 7.4).

After homogenisation in 30 ml of 100 mM tricine (pH 7.5)/ 5 mM thioglycolic acid (TGA),

half the sample was frozen with liquid nitrogen and stored at -80°C. The remaining sample

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Methods

35

was sonicated four times (10 x pulsed, output 5, 50% duty cycle) and protected from the light.

Cell debris was removed by centrifugation at 8,000 x g, 20 min, 4°C (10,000 rpm Beckman

Le-80 ultracentrifuge). Microsomes (in supernatant) were further sedimented by

centrifugation at 105,000 x g for 65 min, 4°C (40,000 rpm, Beckman Le-80 ultracentrifuge).

The resulting microsomal pellet was resuspended in 1.5 ml of 100 mM tricine (pH 7.5)/ 5 mM

TGA using a Pasteur pipette, and homogenized in a chilled glass homogenizer with a Teflon

pestle. 600 µl of the microsomal suspension was transferred into a new Eppendorf tube for

measurement of a CO difference spectrum, the rest brought to 20% glycerol and stored at

-80°C.

3.21. Measurement of CO difference spectra

A difference spectrum of microsomal preparations was measured with a Perkin Elmer

Lambda 800 UV/Visible spectrophotometer with 1 mm light path cuvettes. 600 µl microsomal

suspension were homogenized with 900 µl of 100 mM tricine buffer pH 7.5/ 5 mM TGA and

0.5 % (v/v) emulgen 913 (detergent) and agitated for 15 min. Insoluble debris was removed

by centrifugation (15,000 x g, 15 min, 4°C) and the supernatant was divided into two cuvettes.

A small amount of sodium dithionite (about 1 mg) was added to each sample and mixed by

inverting the cuvette. Microsomal preparations were placed in both the sample and reference

cells of the spectrophotometer. After recording the base line, the content of the sample cell

was carefully gassed with CO for about 1 min and the spectral shift from 420 to 450 nm was

measured.

3.22. Protein determination

Protein concentration was determined according to the method of Bradford (1976) with

known concentrations of bovine serum albumin as standards. This method is based on the

binding of Coomassie brilliant blue G-250 dye (CBB) to proteins with an absorbance

maximum at 595 nm (blue).